Characterization of a highly conserved island in the otherwise divergent Bordetella holmesii and Bordetella pertussis genomes

Abstract

The recently discovered pathogen Bordetella holmesii has been isolated from the airways and blood of diseased humans. Genetic events contributing to the emergence of B. holmesii are not understood, and its phylogenetic position among the bordetellae remains unclear. To address these questions, B. holmesii strains were analyzed by comparative genomic hybridization (CGH) to a Bordetella pertussis microarray and by multilocus sequence typing. Both methods indicated substantial sequence divergence between B. pertussis and B. holmesii. However, CGH identified a putative pathogenicity island of 66 kb that is highly conserved between these species and contains several IS481 elements that may have been laterally transferred from B. pertussis to B. holmesii. This island contains, among other genes, a functional, iron-regulated locus encoding the biosynthesis, export, and uptake of the siderophore alcaligin. The acquisition of this genomic island by B. holmesii may have significantly contributed to its emergence as a human pathogen. Horizontal gene transfer between B. pertussis and B. holmesii may also explain the unusually high sequence identity of their 16S rRNA genes.

FIG. 1.

Comparative genome hybridization of B. holmesii strains to a classical Bordetella DNA microarray. (A) CGH of 12 B. holmesii isolates to a microarray comprising the genomes of B. pertussis Tohama I, B. parapertussis 12822, and B. bronchiseptica RB50. The moving average (with a sliding window of three adjacent values) of the mean log₂(Cy5/Cy3) of 12 B. holmesii genomes is plotted on the x axis. Microarray probes that are represented in the B. pertussis Tohama I genome are arranged on the y axis in B. pertussis Tohama I genome order. The dashed line indicates the fifth percentile of B. pertussis Tohama I hybridization ratios; ratios below the fifth percentile are considered to indicate the lack of B. holmesii hybridization. (B) Probes that hybridized to the B. holmesii genome with a strength comparable to that of the reference and adjacent nonhybridizing probes are shown in detail for individual B. holmesii strains and for B. pertussis Tohama I. ORF and gene designations for selected loci are provided as landmarks. Probes representing insertion sequence elements are also shown. BB2492 is a putative IS3 family transposase found in B. bronchiseptica RB50 but not in B. pertussis. Strain numbers are indicated above the columns. Each row represents one probe in B. pertussis Tohama I gene order. The logarithm of the hybridization ratio [log₂(Cy5/Cy3)] is indicated by the yellow-black-blue color scale. Missing data are represented in gray. The data represented in this figure are available as Table S4 in the supplemental material.

FIG. 2.

Comparison of IUI genomic organizations in B. holmesii, B. pertussis, B. bronchiseptica, and B. avium. Deletions and insertions between species are indicated by gray surfaces. The dashed lines connect the ORFs at the borders of the orthologous sequences. The ORFs are color coded as described in the key. The ORF composition of B. holmesii was deduced from PCR and CGH data, while the representations of B. avium 197N, B. pertussis Tohama I, and B. bronchiseptica RB50 ORFs were based on published genome sequences.

FIG. 3.

LC-MS spectra of B. holmesii supernatants from iron-depleted and iron-replete cultures. (A) LC-MS spectra of iron-depleted (−Fe) and iron-replete (+Fe) B. holmesii supernatants, depicting ions with m/z 405 (corresponding to alcaligin) and with retention times between 0 and 45 min. (B) Fragment ions detected after collisional activation (35% energy) of the peak from (A), with a retention time of 36.99 min. After measurement of the reference standard, the calibration deviated by approximately 1.5 mass units, explaining the difference in m/z values compared to those of the fragment ions as reported in reference . The nomenclature of fragment ions is according to that in reference .

FIG. 4.

Neighbor-joining phylogeny of Bordetella spp. and related β-proteobacteria based on four genes outside the rRNA operon. The unrooted tree is based on 3,559 fully informative nucleotides from an alignment of concatenated sequences of atpD, rpoB, tuf, and rnpB from seven B. holmesii strains, B. pertussis Tohama I, B. parapertussis 12822, B. bronchiseptica RB50, B. avium 197N, B. hinzii BC304, B. trematum DSM11334, and B. petrii SE1111R, and from A. xylosoxidans ATCC 15173 and B. pseudomallei K96243. Bootstrapping values greater than 50% (in 1,000 resamplings) are indicated at branches.

FIG. 5.

Southern blot hybridizations of B. pertussis, B. holmesii, and B. avium with 16S rRNA gene probes. Genomic DNA from each of the three strains was restriction digested with ClaI or NcoI as indicated and hybridized to a B. pertussis-specific probe (left panel) and a broad-range 16S rRNA gene probe (B-16S8F [right panel]) (15). Biotinylated DNA markers are in the first lane, and numbers at the left are fragment sizes (in base pairs).